Parallel evolution and enhanced virulence upon in vivo passage of an RNA virus in Drosophila melanogaster

Abstract Virus evolution is strongly affected by antagonistic co-evolution of virus and host. Host immunity positively selects for viruses that evade the immune response, which in turn may drive counter-adaptations in host immune genes. We investigated how host immune pressure shapes virus populations, using the fruit fly Drosophila melanogaster and its natural pathogen Drosophila C virus (DCV), as a model. We performed an experimental evolution study in which DCV was serially passaged for ten generations in three fly genotypes differing in their antiviral RNAi response: wild-type flies and flies in which the endonuclease gene Dicer-2 was either overexpressed or inactivated. All evolved virus populations replicated more efficiently in vivo and were more virulent than the parental stock. The number of polymorphisms increased in all three host genotypes with passage number, which was most pronounced in Dicer-2 knockout flies. Mutational analysis showed strong parallel evolution, as mutations accumulated in a specific region of the VP3 capsid protein in every lineage in a host genotype-independent manner. The parental tyrosine at position ninety-five of VP3 was substituted with either one of five different amino acids in fourteen out of fifteen lineages. However, no consistent amino acid changes were observed in the viral RNAi suppressor gene 1A, nor elsewhere in the genome in any of the host backgrounds. Our study indicates that the RNAi response restricts the sequence space that can be explored by viral populations. Moreover, our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.


Introduction
Viruses are ubiquitous parasites of cellular life and the most abundant biological entities on earth, predicted to appear in any replicator system (Koonin and Dolja 2013;Iranzo et al. 2016;Krupovic, Dolja, and Koonin 2019).Virus-host coevolution is often understood as an arms race between host defense and viral counterdefense mechanisms that occurs in all host systems (Finlay and McFadden 2006).For example, the interferon response, one of the most potent antiviral mechanisms in mammals, can be antagonized at the transcriptional, translational, or functional level by many different viruses (García-Sastre and Biron 2006).
In insects, RNA interference (RNAi) plays a critical role in antiviral defense, as viral double stranded RNA (dsRNA) is processed by the endonuclease Dicer-2 (Dcr-2) into small interfering RNAs (siR-NAs), serving as guides for the endonuclease Argonaute-2 (AGO2) to mediate viral RNA degradation (Gammon and Mello 2015).Several insect viruses have been shown to suppress this pathway, illustrating how immune pressure exerted by RNAi affects the genetic makeup of a virus (Bronkhorst and van Rij 2014;Guo, Li, and Ding 2019).For instance, the Drosophila C virus (DCV) 1A protein binds double-stranded RNA (dsRNA), interacts with Dcr-2, and inhibits Dcr-2 nuclease activity (van Rij et al. 2006;Nayak et al. 2018), whereas proteins from two distinct RNA viruses, the cricket paralysis virus 1A protein and the nora virus VP1 protein bind and inhibit AGO2 function (Nayak et al. 2010(Nayak et al. , 2018;;van Mierlo et al. 2012van Mierlo et al. , 2014)).
Upon infection, RNA viruses create swarms of closely related within-host genotypes, often referred to as viral quasispecies (Lauring and Andino 2010), that contribute to the adaptability of a viral population to changing environments (Lauring, Frydman, and Andino 2013;Duffy 2018).They are the result of the high mutation rate of the viral RNA-dependent RNA polymerase (RdRP) and the large population sizes of RNA viruses, which can be as high as 10 12 infectious particles in an infected host (Moya, Holmes, and González-Candelas 2004;Duffy, Shackelton, and Holmes 2008).Most mutations in this diverse virus population will be deleterious, some neutral, and very few beneficial (Hughes 1999;Acevedo, Brodsky, and Andino 2014), and the frequency of these variants will fluctuate according to evolutionary forces, such as the positive and purifying selection imposed by the host immune system.New virus variants that are capable of escaping immune surveillance have an evolutionary advantage and will likely take over the ancestral population.In accordance, plants with a more restrictive immune system have been shown to drive faster evolution of turnip mosaic virus (Navarro et al. 2022).
In this study, we assessed to what extent selection pressure by the immune system drives virus population evolution.We used as a model system the fruit fly Drosophila melanogaster infected with its natural pathogen DCV, a single-stranded positive-sense RNA virus of the Dicistroviridae family (genus Cripavirus).The virus was serially passaged in wild-type flies and in fly mutants in which Dcr-2 was either overexpressed or inactivated.Afterwards, the evolved virus populations were characterized using next-generation sequencing (NGS) in combination with phenotypic assays for virus replication and host survival.We found that the increase in the number of polymorphisms during passaging was most pronounced in Dicer-2 knockout flies.However, the RNAi response had little effect on the accumulation of specific amino acid changes in viral proteins.Instead, we observed parallel evolution of mutations in the VP3 capsid protein in all viral lineages, which markedly enhanced viral replication and virulence in vivo.

Experimental DCV evolution increases virulence
To investigate the effect of the host immune system on DCV evolution, we performed an experimental evolution study in which a parental DCV stock was serially passaged over three fly genotypes with different RNAi activity (Fig. 1A).Specifically, we used wild-type flies (w 1118 , hereafter WT), flies with a frameshift mutation in Dcr-2 resulting in a premature stop codon (Dcr-2 L811fsX (Lee et al. 2004), hereafter Dcr-2-KO), and flies overexpressing a Dcr-2 transgene controlled by the UAS upstream activation sequence and a Tubulin-Gal4 driver (hereafter Dcr-2-OE).As expected (Galiana-Arnoux et al. 2006;Spellberg and Marr 2015), Dcr-2 expression correlated with survival after DCV infection, with Dcr-2-KO flies having the shortest life span and Dcr-2-OE flies the longest (Fig. 1B).
For each host genotype, we allowed virus populations to independently evolve during ten serial passages, with five independent lineages per genotype.The parental DCV stock was cultured in Drosophila S2 cells, and its genetic diversity had been reduced by three rounds of serial dilution prior to the experimental evolution study.To prevent population bottlenecks, flies were infected by intrathoracic injection with a high viral inoculum of 10 4 median tissue culture infectious doses (TCID 50 ), and virus titers were measured after each passage to normalize the inoculation of naive flies for the next passage (Fig. 1C).With the exception of passages 3 and 5, where we observed significantly higher viral titers in Dcr-2-KO than in WT flies (P < 0.05, t-test, Fig. 1C), the differences in viral titers between genotypes were generally small and not consistent along passages, likely because flies were harvested at the plateau of peak titers.
To examine the virulence of the evolved virus populations, we performed survival assays after inoculation of WT flies with 10 4 TCID 50 of the parental stock and virus stocks harvested after passages 1, 5, and 10 (P1, P5, P10; Fig. 2A-D).P10 virus populations from all lineages of all host genotypes induced higher mortality in WT flies than the parental stock (Log-rank test, P < 0.0001; hazard ratios > 3, Supplementary Table S1), with mean survival times decreasing from 5.5 days for the parental stock to 3.4 days for the P10 virus populations (average of all genotypes and lineages).The mean survival time of WT flies decreased from P1 to P5 for virus populations from all genotypes (paired t-test, P < 0.005) and further decreased from P5 to P10 only for virus populations from WT flies (paired t-test, P = 0.006) (Fig. 2D).
We next analyzed whether these survival phenotypes correlated with changes in replication kinetics of the evolved virus populations.To test this, we inoculated WT flies with the parental, P1, and P10 virus stocks and determined viral RNA levels at 12 h post-infection (hpi) (Fig. 2E).We observed a large variation in viral RNA replication between the independent lineages of all genotypes at P1, with the parental stock accumulating 3.4 × 10 5 RNA copies and the P1 viruses ranging from 4.2 × 10 4 to 3.9 × 10 7 RNA copies.Irrespective of the genotype, all lineages of the P10 virus populations reached higher viral RNA levels (range 2.8 × 10 6 to 2.2 × 10 8 RNA copies) than the parental stock (nested ANOVA with significant effect of genotypes on the viral RNA level: P < 0.001, followed by Tukey's HSD test with adjusted P-value: P-adj.< 0.0001).This phenotype was maintained when the infection was performed orally (Supplementary Fig. S1), indicating that our experimental evolution scheme did not lead to loss of infectivity via the presumed natural infection route.Among genotypes, P10 virus populations from Dcr-2-KO flies reached the highest RNA loads, compared to those evolved in WT and Dcr-2-OE flies (P-adj.= 0.0003 and P-adj.= 0.0468 Tukey's HSD test), where those evolved in Dcr2-OE flies reached higher RNA loads compared to WT (P-adj.= 0.0374 Tukey's HSD test) (Fig. 2E).Together, these results indicate that in vivo serial passage increases replication kinetics and virulence of the evolved virus populations.

Ongoing accumulation of polymorphisms
We next investigated how the virus populations evolved through the serial passage experiment by NGS.Viral genomes were sequenced to high coverage (average coverage of 127,542, Supplementary Fig. S2), allowing us to confidently call low-frequency mutations, as the median Phred sequencing quality score for the mutation calls was 39, corresponding to an error rate of 0.0001.We observed increasing numbers of mutations during passaging with a mean of 381 polymorphic sites in the evolved virus populations after ten passages, 116 more than in the parental stock (Fig. 3A).Between 179 and 499 single-nucleotide variants (SNVs) per evolved population occurred at a frequency below 1 per cent, and between five and twenty-one SNVs at a frequency higher than 1 per cent (Fig. 3B).The number of polymorphic sites increased significantly through the serial passage in Dcr-2-KO and Dcr-2-OE flies (P < 0.05, paired t-test, Fig. 3A).This increase was most pronounced in virus populations from Dcr-2-KO flies, which accumulated a mean of 444 polymorphic sites at passage 10 (Fig. 3A), which was significantly different from virus populations from WT and Dcr-2-OE flies (P < 0.02, t-test).Yet, the vast majority of polymorphic sites were low-frequency mutations (median: 0.1 per cent), and very few SNVs increased in frequency or reached fixation (Fig. 3B).
We next analyzed virus population diversity over time and found that the mean nucleotide diversity did not change significantly over time in any of the host genotypes, nor were there significant differences in population diversity between genotypes (P > 0.05, mixed ANOVA, Supplementary Fig. S3A).Mean nucleotide diversity was significantly higher in coding than in non-coding regions of the viral genome (P < 0.01, mixed ANOVA, Supplementary Fig. S3A).Specifically, the gene encoding the viral VP3 protein displayed a significant increase in nucleotide diversity compared to the mean diversity of the entire coding region (P < 0.05, paired t-test, Supplementary Fig. S3B).

Moderate host genotype-specific adaptation in Dicer-2 mutants
We next assessed the shared and unique SNVs across genotypes, first considering SNVs with frequencies ≥ 0.01 per cent, and found a similar number of unique SNVs between virus populations from WT and Dcr-2-OE flies (n = 813 and 910, respectively), both having less SNVs than virus populations from Dcr-2-KO flies (n = 1,189) (Fig. 3C).A 15.7 per cent of the SNVs were shared between virus populations from all three fly genotypes, of which 41.07 per cent were non-synonymous mutations.When restricting the analysis to high-frequency SNVs (≥ 10 per cent), the fraction of SNVs shared by the virus populations from all three fly genotypes increased to 21.3 per cent, all of which were non-synonymous mutations (Fig. 3D).
To understand if the observed mutation patterns are the result of host-specific adaptation, we conducted a permutation test, as described by Bons et al. (Bons et al. 2020).This test assesses whether SNVs occur at higher rates in specific genotypes than expected under a null hypothesis that SNVs are equally likely to occur in each genotype.For virus populations from WT and Dcr-2-OE flies, we did not find significant differences between the expected and observed number of SNVs.In contrast, 131 SNVs were unique to virus populations from Dcr-2-KO flies, which was significantly different from the expected number by random allocation (n = 89; P < 0.001, Supplementary Fig. S4).Together, these data indicate that viral populations are predominantly shaped by host-genotype-independent, parallel evolution of shared SNVs as well as moderate host genotype-specific adaptation in the absence of a functional RNAi response.

Deletions in homopolymeric uridine tracts
To explore the evolution of the viral populations in more detail, we visualized the SNVs across the DCV genome in a heatmap (Fig. 4A).We observed some SNVs that were already present in the parental stock and were retained in the viral populations throughout the experiment in most of the lineages (nt 329, 504, 2686, 2736, 7082, 7458, 8089, and 8526), while others appeared de novo during the experiment in different lineages (e.g. nt 331, 1026, 4758, 6263, and 7557).Strikingly, we found a ubiquitous deletion of a uridine in a poly(uridine) tract at position 5765-5774, which gives rise to a frameshift and a premature stop codon in the RdRP gene.This deletion was also present in the parental stock and it was retained at low, yet fairly constant frequency throughout the experiment in all fifteen lineages (around 4 per cent, Supplementary Table S2).Likewise, another uridine deletion in a tract of eight uridines at position 276-284 in the 5 ′ untranslated region (UTR) occurred in all fifteen lineages with a constant frequency of around 3 per cent (Supplementary Table S2).To control for possible sequencing artifacts, we evaluated if the two uridine deletions were located at the edges of the sequence reads containing them, as there is lower confidence in the base calling at read edges.However, in only 7 per cent of reads the deletions were located in proximity to the read edges (within five nucleotides).Furthermore, the mean sequencing quality score in the uridine tracks and the adjacent positions was above forty both in reads with and without deletion, corresponding to an error rate of 0.0001 (mean quality scores across uridine tracks: 40.1; 10 per cent-quantile: 38.2, 90 per cent-quantile: 41.0; Supplementary Table S3).Further analysis suggested that such deletions also occur in other, published DCV NGS datasets, and we suggest that the viral RdRP is prone to slippage on homopolymeric uridine tracts and that there is purifying selection against the occurrence of these tracts in RNA viruses (Supplementary text S1, Supplementary Table S4

Parallel evolution is driven by positive selection on the capsid protein
Thirteen SNVs became dominant (frequency > 50 per cent) after ten passages, most of which changed the amino acid sequence of the capsid proteins in a host genotype-independent manner (Figs.3D and 4B).Of note, two high-frequency SNVs appeared to be host genotype specific: 7118C became dominant only in WT flies (at a frequency of 99 per cent in one lineage and above 20 per cent in two others) and 6845 G became dominant in two lineages from Dcr-2-OE flies.However, the majority of the dominant SNVs were located at codon 95 of the capsid VP3 gene (nt 7566-7567), where the tyrosine codon (Y95) in the parental stock was replaced in fourteen out of fifteen lineages (Fig. 5A).After one passage, the Y95 frequency already declined below 60 per cent in three out of five lineages in Dcr-2-OE flies, whereas it remained present at frequencies above 90 per cent in four and three lineages in Dcr-2-KO and WT flies, respectively.After five passages, Y95 was undetectable in twelve out of fifteen lineages, and at P10, Y95 was only retained in lineage D from Dcr-2-OE flies (at a frequency of 6 per cent) and in lineage A from Dcr-2-KO flies, which was the only lineage in which Y95 remained dominant.Interestingly, in the latter lineage, two other capsid mutations, VP3 P92T and VP1 N114S, were fixated instead.
Strikingly, from all nine possible SNVs that can be formed from the parental UAU codon coding for Y95, five were found in the evolved virus populations, coding for aspartic acid, asparagine, cysteine, serine, and histidine.The emergence of these variants did not correlate with the host genotype in which the virus populations had evolved.The remaining four possible codons were not detected, two of which being stop codons, one coding for a synonymous substitution, and one coding for phenylalanine, the amino acid most similar to tyrosine.Overall, these results suggest that the presence of a benzene ring at residue 95 of VP3 is detrimental to virus infection in flies.
To confirm the selection forces statistically, we computed the population nucleotide diversity per synonymous (piS) and non-synonymous (piN) site for each viral gene separately (Supplementary Fig. S5).There were significant main effects of viral gene, site type (synonymous and non-synonymous), and passage number (P < 0.001, mixed ANOVA), and significant interactions between viral gene and site type (P < 0.0001) and between gene and passage number (P < 0.05), but no significant effects of host genotype on the population nucleotide diversity (P > 0.05).We found clear indication of positive selection (piN-piS > 0) in VP3 across all passages (P1: P-adj.= 0.01;

An exposed amino acid in the capsid is responsible for environment-specific fitness gains
We hypothesized that the VP3 mutations were responsible for the observed fitness gains of the passaged virus populations (Fig. 2E).
To test this, we selected P10 lineages with one of the fixated mutations (frequency ≥ 90 per cent), performed a serial dilution to remove minority variants, and analyzed growth kinetics in the Drosophila S2 cell line and in WT flies inoculated with a low dose (multiplicity of infection of 0.1 and 100 TCID 50 , respectively).All variants efficiently replicated in S2 cells, but the parental stock showed higher replication kinetics than the other variants (unpaired t-test, P < 0.01, Fig. 5B).In contrast, the parental stock scarcely replicated in adult flies at this low inoculum, whereas the evolved variants replicated efficiently in vivo (unpaired Welch's t-test, P < 0.01, Fig. 5C).These results suggest that Y95 may be beneficial for DCV replication in cell culture, but detrimental for replication and virulence in vivo.
To investigate the effects of Y95 on the structural stability of the virion, we modeled the effect of the fixated mutations on the structure of the virus particle.We used the crystal structure of cricket paralysis virus (CrPV) (Tate et al. 1999), a related dicistrovirus which shares 60 per cent sequence identity with the DCV capsid polyprotein and 79 per cent identity within the region of interest.Given the high identity between these two proteins, it is possible to model the variants in CrPV capsid to predict their probable implications on the DCV capsid.The capsid polyprotein is a large assembly of 240 subunits, composed of copies of four proteins (VP1, VP2, VP3, and VP4) (Fig. 6A).The region of interest is located in a loop close to the protein-protein interface between VP2 and VP3 (Fig. 6A, C-E).While this region is conserved across related dicistroviruses that share more than 50 per cent sequence identity with the DCV capsid, the residues at positions 92 and 95 are relatively poorly conserved (Fig. 6B).Moreover, given their location in the loop, these residues are predicted to be solventexposed, suggesting that they may not be involved in maintaining the structural integrity of the capsid.Indeed, we do not find drastic differences in the predicted folding free energy of the capsid structure between the parental Y95 and its mutants (ranging from 0.71 to 3.33 kcal/mol; Supplementary Table S7), suggesting that the observed fitness gains cannot be explained by structural effects of the acquired mutations.

Discussion
In this study, we have explored the effect of immune pressure on virus evolution using Drosophila as a model host.We passaged DCV in flies differing in Dcr-2 activity, hypothesizing that variations in RNAi immune pressure would affect virus evolution at the phenotypic and genotypic level, for instance by removing selection from the viral 1A protein that interferes with the host RNAi pathway.However, the effect of the RNAi machinery on DCV evolution was moderate.No specific polymorphisms accumulated in the DCV 1A gene in virus populations from Dcr-2-KO flies, nor in the other genotypes.However, we did observe that virus populations from Dcr-2-KO flies accumulated more polymorphisms than populations from Dcr-2 expressing flies, in line with previous observations (Mongelli et al. 2022), suggesting that the absence of immune pressure releases selective constraints, reducing purifying selection and allowing virus populations to further explore the mutational landscape.
It will be of interest to use our experimental approach to study the evolution of viruses that differ in their mode of RNAi suppression.Although DCV 1A interacts with several proteins including Dicer-2 (Nayak et al. 2018), it relies on its dsRNA binding activity to suppress RNAi (van Rij et al. 2006).As such, DCV may be under different selection pressure than viruses that suppress RNAi via direct protein-protein interactions, such as CrPV and Nora virus (Nayak et al. 2010(Nayak et al. , 2018;;van Mierlo et al. 2012van Mierlo et al. , 2014)).It will likewise be interesting to study DCV evolution in flies bearing alleles of the pastrel restriction factor, which has strong effects on viral replication and virulence and may encode a protein that directly interacts with viral proteins (Magwire et al. 2012).
Even though we observed some host genotype-specific effects on virus evolution, the strongest evolutionary pattern was shared among virus populations from all host genotypes, suggesting parallel and reproducible evolution.Specifically, the parental tyrosine at position 95 of VP3 was substituted with either one of five different amino acids, resulting in strongly enhanced replication and virulence in vivo.The predictability and reproducibility of evolutionary trajectories across independent populations has been a long-standing problem (Gould 1989).Examples of parallel evolution exist, for example, a mutation giving rise to antibiotic resistance was observed in multiple isolates of Escherichia coli independently (Coolen et al. 2021), and specific drug resistanceassociated mutations appeared repeatedly in human immunodeficiency virus-1 (HIV-1) in individuals receiving antiretroviral therapy (Crandall et al. 1999;Beerenwinkel et al. 2005;Flynn et al. 2015).Likewise, the same subset of mutations has arisen independently in HIV-1 in two different cell lines during a long-term experimental evolution study (Bertels et al. 2019).In contrast, in the long-term evolutionary study of Lenski et al. (Lenski et al. 1991), the phenotype allowing E. coli to use citrate in aerobic conditions occurred in multiple independent lineages, but the underlying molecular mechanism was different (Blount, Borland, and Lenski 2008).In our experiment, VP3 gene mutations occurred in parallel in all virus lineages, concomitant with a fitness gain and higher virulence in vivo.These mutations were fixated in fourteen out of fifteen lineages independently, indicating that loss of Y95 is a highly reproducible, yet unpredictable process, as five different variants have taken over the population.
Another instance of polymorphisms occurring in all lineages were deletions of a single uridine in homopolymeric tracts of at least six uridines in the 5 ′ UTR and in the RdRp gene.These uridine deletions also occurred in NGS data of DCV from other studies, and we propose that they are caused by RdRP slippage on homopolymeric uridine tracts.In contrast, thymidine deletions did not occur in NGS data from a DNA virus, herpes simplex virus 2, likely as a result of the proofreading activity of its viral DNA polymerase.In agreement, E. coli strains lacking mismatch repair mechanisms show increased numbers of deletions in simple sequences repeats (Kumar and Nagarajaram 2012).
It is well described that insertions and deletions preferentially occur in homopolymeric tracts in viral genomes (Domingo 2020).Some viruses make use of this feature, such as viruses of the Potyviridae family that use transcriptional slippage to insert an adenosine in a specific GAAAAAA sequence in viral transcripts to induce a frameshift and expression of an alternative viral protein (Olspert et al. 2015).Likewise, the measles and Sendai viruses undergo a slippage event that adds an extra guanidine in transcripts coding for the P protein, creating a frameshift transcript that codes for the V protein involved in antagonizing host immunity (Atkins et al. 2016).However, transcriptional slippage also occurs in picornaviruses on homopolymeric stretches of more than six uridines and these sequences are underrepresented in their genome, suggesting that there are also fitness costs associated with poly(uridine) tracts (Stewart et al. 2019).The deletion in the RdRP gene in DCV introduces a premature stop codon in the RdRP and likely results in the production of defective viral genomes (DVGs).DVGs have been reported in many virus families and may affect pathogenesis via different mechanisms, like interfering with antiviral immunity or facilitating viral persistence (Vignuzzi and López 2019).Whether DCV likewise produces DVGs and their involvement in the infection cycle remains to be established.
We found that the parental DCV stock replicated at low levels in flies receiving a low inoculum, but that it efficiently replicated in cell culture.This suggests that the parental virus is unfit because it lacks a basic function that is essential in vivo, but not in cell culture.Considering that the virus replicates efficiently in cells, we expect cell entry not to be impaired.Using structural models, we predicted that Y95 substitutions do not affect virus particle stability and that the residue is solvent exposed.We hypothesize that the observed phenotype is due to immune recognition of the parental stock containing Y95 in VP3, and escape thereof by mutants in VP3.This scenario is not unanticipated, as a single-nucleotide substitution has been found to mediate immune evasion before.For example, blood clearance of alphaviruses like chikungunya virus is mediated mainly by resident macrophages in the liver, but a single amino acid substitution in the E2 glycoprotein disrupts this process and increases virus dissemination (Carpentier et al. 2019).We speculate that the parental virus is recognized by an immune receptor to mediate virus clearance, thus explaining the differences between in vitro versus in vivo replication.
To conclude, we have found that elimination of RNAi-based immune pressure during virus evolution led to a higher accumulation of polymorphisms, perhaps due to a reduction in purifying selection.Moreover, we have observed how a single amino acid change in the capsid of an RNA virus can have dramatic environment-specific effects on the growth rate.Our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.

Fly strains and husbandry
Flies were maintained at 25 ∘ C in standard fly food.Eggs were treated with bleach and, subsequently, flies were treated with tetracycline for two fly generations, as described before (Merkling and van Rij 2015).Absence of Nora virus, DCV, Drosophila X virus, and cricket paralysis virus was confirmed by RT-PCR using random hexamers for cDNA synthesis and the following primers for PCR: NoraFor, ATGGCGCCAGTTAGTGCAGACCT; NoraRev, CCTGTTGTTCCAGTTGGGTTCGA; DCVFor, AAAATTTCGTTTTAGCCCAGAA; DCVRev, TTGGTTGTACGTCAAAATCTGAG; DXVFor, TCGGAAGAACCAAAAGGATG; DXVRev, GTCCTCTCCACGCACTCTTC; CrPVFor, ACGAGGAAGCAACTCAAGGA; CrPVRev, GAGCCCGCTGAGATGTAAAG. Absence of Wolbachia in the fly stocks was confirmed by PCR using primers wspFor TTTGCAAGTGAAACAGAAGG, wspRev GCTTTGCTGGCAAAATGG.Isogenized Dicer-2-KO (Dcr-2 L811fsX ) have been described before (Mongelli et al. 2022).Flies overexpressing Dicer-2 were generated by crossing UAS-Dicer-2 virgin females (VDRC ID 60,008, (Dietzl et al. 2007)) with Tubulin-Gal4/TM3 Sb male flies, after which flies lacking the Sb marker were selected from the F1 offspring.The w 1118 flies were used as wild-type.Two to fiveday-old female flies were used for the serial passage and the RNA extractions.

Virus stock and titration
The parental DCV stock (Charolles strain) was produced in Drosophila S2 cells, which were maintained in Schneider's medium supplemented with 10 per cent fetal calf serum and 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco) medium at 27 ∘ C. The stock was bottlenecked through three rounds of serial dilutions, by selecting the highest dilution that still showed cytopathic effects.Subsequently, a T25 flask of confluent S2 cells containing 5 ml of medium was infected with the bottlenecked virus.After 72 h, the supernatant was collected, centrifuged at 300 g for 5 min, aliquoted, and stored at −80 ∘ C. Titers were determined by endpoint dilution in 96-well plates, as described before (Merkling and van Rij 2015), and expressed as median tissue culture infectious dose (TCID 50 ), calculated with the Reed-Muench method.

Experimental evolution
Females of each genotype were intrathoracically inoculated with 10,000 TCID 50 of DCV in 50 nl of 10 mM Tris-HCl, pH 7.3 using a Nanoject II microinjector (Drummond Scientific).For each of the five lineages per genotype, forty flies were inoculated.For the first passage, flies were inoculated with the parental stock and from P1 onwards, all fifteen lineages were kept independently.After each passage, flies were harvested for preparation of virus stocks.Pools of ten flies were homogenized in 220 μl of PBS, using 1 mm silica beads in a Precellys homogenizer two times for 10 s, centrifuged at 16,000 g for 10 min to discard fly debris, and the supernatant was transferred to fresh tube, aliquoted, stored at −80 ∘ C, and titrated.For RNA extraction and NGS library of the parental stock, 1 ml of RNA-Solv (Omega) was added to 100 μl of virus stock, while for the viral lineages, pools of five flies were homogenized in 1 ml of RNA-Solv (Omega) using 1 mm silica beads in a Precellys homogenizer.

Virus infections and survival assays
To study virulence, 10,000 TCID 50 of all lineages from P1, P5, P10, and the parental DCV stock were injected separately into thirtyfive female w 1118 flies.Survival was checked daily and analyzed using the Kaplan-Meier estimator.Early replication was assessed after inoculation of two to five-day-old female w 1118 flies with 1,000 TCID 50 of all lineages of P1, P10, or the parental stock.Three pools of five flies per lineage were collected at 12 hpi and processed for RT-qPCR.For the oral infections, thirty female w 1118 flies were inoculated with 1,000 TCID 50 by intrathoracic injection, placed in tubes for 3 days, after which these flies were discarded and thirty naive female w 1118 flies were added to the contaminated vial.Three pools of five flies were collected at 48 hpi and processed for RT-qPCR.
To analyze replication of the different capsid variants, P10 virus lineages containing the variant of interest at a frequency ≥ 90 per cent were bottlenecked by serial dilution.Supernatant from the last well showing cytopathic effect in an end-point dilution was used to grow stocks in a T25 flask, followed by titration and Sanger sequencing to confirm the presence of the mutation.A total of 100 w 1118 female flies were inoculated with 100 TCID 50 of each viral stock, and three pools of five flies per variant were collected at the given time points and processed for RT-qPCR.

RT-qPCR
Total RNA was extracted, reverse transcribed using the Taq-Man reverse transcription kit (Thermo Fisher) using oligo (dT) as a primer and quantitative PCR analysis was performed using the GoTaq qPCR SYBR master mix (Promega) on a LightCycler 480 instrument (Roche), according to the manufacturers' recommendations.DCV was quantified using primers DCVFor (TCAAGAAAAGTTGCGTGGGT) and DCVRev (CAGAGCGTCCTTG-GAGAGTG), and expression was normalized to expression of Ribosomal protein 49, amplified using primers Rp49For (ATGACCATC-CGCCCAGCATAC) and Rp49Rev (CTGCATGAGCAGGACCTCCA).A standard curve of 10-fold dilutions of plasmid pAc5.1-V5-His-A(Invitrogen) containing the DCV ORF-2 sequence was used to convert Ct values to genome copy numbers.

NGS data processing
NGS data were analyzed using a custom computational workflow (Supplementary Fig. S6).For processing the raw read data, the bioinformatics pipeline V-pipe (Posada-Céspedes et al. 2021) was integrated into the workflow.Burrows-Wheeler Alignment Tool (BWA-MEM) (Li and Durbin 2009) was used to align reads from the parental stock to the DCV EB reference strain that contains complete UTR sequences (GenBank accession number: NC_001834.1).Bcftools was used to generate a consensus sequence from the parental sequence, which was then used to align the raw reads from the evolutionary samples from passages 1, 5, and 10 using BWA-MEM.Mutations with respect to the parental stock consensus sequence were called using Short Read Assembly into Haplotypes (Zagordi et al. 2011) with default parameters (posterior threshold of 0.9, alpha 0.1 and shift 3).The posterior threshold was chosen as conservative to ensure that only mutations of high confidence are included in the subsequent analyses.Moreover, the Strand Bias Filter (McElroy et al. 2013) was applied to filter out further variation of low confidence.Phred sequencing quality scores for mutation calls were accessed using pysamstats (https://github.com/alimanfoo/pysamstats). Further, to evaluate the differences of our parental stock to its ancestor, the Charolles strain, the parental stock sample was processed with respect to the Charolles reference sequence (GenBank Accession Number: MK645242.1).An adapted version of vcf_annotator (https://github.com/rpetit3/vcf-annotator) was used to annotate non-synonymous mutations.Population nucleotide diversity and number of polymorphisms were computed considering only mutations with a minimum frequency of 0.0001.SNPGenie's within-pool analysis was used in the default settings and minimum frequency 0.0001 (Nelson, Moncla, and Hughes 2015) to compute the population nucleotide diversity per synonymous (piS) and non-synonymous (piN) site.

Permutation test for genotype-specific adaptation
A permutation test as described before (Bons, Bertels, and Regoes 2018) was used to test for host genotype-specific adaptation.Mutations that reached the frequency threshold of 0.0001 in at least one evolutionary line were considered for this analysis.For each mutation, the number of lineages containing the mutation was counted.Then, each mutation occurrence was randomly reallocated to a lineage by using the number of mutations that occurred in each lineage as weights.In this manner, 1,000 samples were drawn.The observed number of mutations that were unique or shared among genotypes was then compared to the number expected from the permutation samples, and a two-sided test was used to assess the difference.For each category, P-values were computed as p = 1 − 2 | 1 n s n s ∑ i=1 1 {x i >x obs } − 0.5| where n s is the number of samples drawn, x i is the i-th random realization and x abs is the observed number of mutations in the respective category.

Randomization test
A randomization test was performed to test whether the observed number of two consecutive phenylalanine (FF) residues encoded as 'TTTTTT' was different from the expected number based on random mixing of the TTT and TTC codons.For each viral genome, the total number of FF occurrences, the number of FF encoded by TTTTTT (n TTTTTT ), and the occurrences of the TTC and TTT codon were counted.For each viral genome, n s = 1000 samples were drawn, by randomly pairing the TTC and TTT codons based on their codon frequencies.To test whether the number TTTTTT encodings from the randomization exceeds the observed number, a two-sided test was performed.P-values were computed

Quality analysis of deletion calls
To evaluate the quality of reads supporting the two uridine deletions at positions 276-284 and 5765-5774, an additional quality control was performed on the alignment files of all samples.The mean position-wise base calling quality scores were assessed in the homopolymeric regions of the two deletions and the values were compared between reads with deletions to the values of reads without the deletions.In addition, the proportion of reads where the deletion track was located within a range of five nucleotides from the read edges was quantified.

Figure 1 .
Figure 1.Experimental evolution of DCV in flies differing in their RNAi response.(A) A parental stock of DCV Charolles strain was serially passaged in wild-type flies (w 1118 ; WT), Dicer-2 knockout flies (Dcr-2-KO), and flies overexpressing Dicer-2 (Dcr-2-OE) for ten fly generations with five independent lineages per genotype.Flies were inoculated by intrathoracic injections and titers were determined after every passage.Evolved virus populations were characterized phenotypically using growth curves and survival assays and genotypically by NGS.(B) Survival curves of WT, Dcr-2-KO, and Dcr-2-OE flies inoculated with 1,000 TCID 50 of DCV.Two to five-day-old female WT flies were inoculated intrathoracically (n = 30) with the parental stock and survival was monitored daily.(C) Viral titers of all lineages at each viral passage.Bars represent means and SD of the five viral lineages.Asterisks indicate P < 0.05, t-test.Fly and virus icons were retrieved from BioRender.TCID 50 , median tissue culture infectious dose.

Figure 2 .
Figure 2. Increased virulence of evolved virus populations independent of the host genotype.(A-C) Survival curves of WT flies inoculated with 10,000 TCID 50 of the parental DCV stock (P0) and virus populations from the indicated host genotypes at passages 1, 5, or 10.Two to five-day-old female WT flies (n = 30) were inoculated intrathoracically and survival was monitored daily.Results are shown as means and SEM of all five lineages for virus populations from (A) WT, (B) Dcr-2-OE, and (C) Dcr-2-KO flies.Data for the parental stock are shown as means and SEM of four independent experiments.Results from Cox regressions are shown in Supplementary Table S1.(D) Difference of the mean survival time for each virus stock relative to the parental stock.Dashed lines connect the values of each lineage.Asterisks indicate significance: ** P < 0.01; ***, P < 0.001, paired t-test.(E) Early replication kinetics of parental and evolved virus populations.WT flies were inoculated with 1,000 TCID 50 of each virus stock and three groups of five flies were collected at 12 hpi.Viral RNA copy numbers were quantified by RT-qPCR.The dashed line shows the RNA copies of the parental stock.Results are shown as means and SD of three replicates.Viral RNA levels of the P10 virus populations and the parental stock were compared using a nested ANOVA followed by Tukey's HSD test for multiple comparison.Adjusted P-values of Tukey's HSD test are indicated with asterisks: *P < 0.05; ***P < 0.001.

Figure 3 .
Figure 3. Increasing genetic variation during serial passage.(A) Number of polymorphic sites of the virus populations from WT, Dicer-2-OE, and Dicer-2-KO flies across passages.Data are shown as means and SEM across the five lineages for each genotype.The dashed line marks the number of polymorphic sites in the parental stock with respect to the Charolles reference sequence (GenBank Accession Number: MK645242.1).(B) Frequencies of single-nucleotide variants (SNVs) in the indicated bins for virus populations from the indicated genotypes.Error bars indicate 95 per cent confidence intervals based on the bootstrap distribution.(C-D) Venn diagram of the number of unique and shared SNVs between virus populations from WT, Dicer-2-OE, and Dicer-2-KO flies.Per genotype, SNVs from all lineages and passages with a minimum observed frequency of 0.01 per cent (C) or 10 per cent (D) were combined.The ratios in (C) indicate the number of non-synonymous mutations to all mutations.Asterisks in (D) indicate non-synonymous mutations.

Figure 4 .
Figure 4. Parallel evolution of virus lineages.Heatmap of SNV frequencies of all SNVs (A) or non-synonymous SNVs (B) for virus populations from WT, Dicer-2-OE, and Dicer-2-KO flies.The x-axis represents genome positions relative to the DCV EB reference strain (NC_001834.1).Only SNVs that occurred in at least one sample with an observed frequency ≥ 0.01 are shown.Deletions were excluded from the analysis in panel (B).A schematic representation of the DCV genome is shown below the heatmaps.

Figure 5 .
Figure 5.A capsid mutation increases in vivo viral fitness.(A) Frequencies of the codon coding for amino acid 95 of VP3 in DCV populations from the indicated host genotypes at passages 1, 5, and 10.UAU represents the parental codon coding for tyrosine (95Y).One variant retained the 95Y residue, but contained a substitution in its vicinity (P92T).(B) Growth curves of individual variants in Drosophila S2 cells infected at an MOI of 0.1.Variants were named according to the host genotype, lineage, and passage number.P0 is the parental virus.Three wells were collected per time point for viral RNA quantification by RT-qPCR.Viral RNA levels are normalized to the 0 hpi time point and presented as geometric mean and SD.(C) Growth curves of individual variants in WT flies after inoculation with 100 TCID 50 of each variant.Three pools of five flies were collected per time point for viral RNA quantification by RT-qPCR.Viral RNA levels are normalized to the 0 hpi time point and presented as geometric mean and SD.Differences in log10-transformed viral RNA levels between the parental and evolved lineages at 96 hpi were analyzed by t-tests (B) and Welch's unequal variances t-tests (C).**P < 0.01.Data for lineage OEa10 in (C) did not follow a normal distribution and a Mann-Whitney U-test was performed instead (P = 0.05).

Figure 6 .
Figure 6.Structural analysis of capsid mutations selected during serial passage.(A) The CrPV capsid structure (PDB: 1B35) is shown with VP1 in blue, VP2 in green, VP3 in yellow, and VP4 in red (bottom).View at the fivefold symmetry axis of the capsid in cartoon representation with Tyr95 and Pro92 highlighted as spheres (top).(B) Multiple sequence alignment of the capsid of DCV and related viruses in the VP3 region of interest.Accession numbers are followed by their amino acid sequence identities to the DCV reference sequence (NP_044946.1).Completely conserved residues are highlighted.(C) The parental residues Tyr95 and Pro92 are located in a solvent-exposed loop connecting two beta strands.Amino acids surrounding Tyr95 and Pro92 within a distance of 6 Å are shown as sticks.(D) A similar view showing the Tyr95 to Ser95 substitution selected upon serial passage.(E) A marginally stabilizing mutant Pro92Thr (−1.79 kcal/mol) is shown.Proximity of the loop to VP2 (green) at a distance of 4.7 Å is depicted in panels (C-E).

1
{x i >n TTTTTT } − 0.5| where x i denotes the number of TTTTTT realizations in the i-th randomization.